To induce immunotolerance, chicken embryos were inoculated with BSA on embryonic incubation day (EID) 20. All hatched chickens were challenged with BSA four times at 10-day intervals, beginning at 3 weeks of age. Serum anti-BSA antibodies were analyzed and our results show that immunotolerance was obtained in the group micro-injected with 1 mg of BSA. We administered BSA labeled with 2,4-dinitrophenyl (DNP) into blood vessels on EID 20 and detected the distribution of BSA by immunohistochemistry. Our results show that DNP-BSA was located in the bursal cortex, thymus and spleen. PBMCs were separated on the seventh day after the fourth immunization to determine the effect of treatments on lymphocytes and the percentage of CD4(+) T cells among the CD3(+) T cell population. Results from these experiments show that there was an influence on T lymphocyte proliferation, with the number of CD4(+) T cells in the tolerance group significantly increased. To determine whether tolerance was induced in B cells, 2,4,6-trinitrophenyl coupled to BSA (TNP-BSA) was inoculated into birds, followed by detection of anti-TNP antibodies in the serum. Immunological tolerance in B cells was not observed following these experiments. The results from our study demonstrate that immunological tolerance was induced in T cells when 1 mg of BSA was micro-injected on EID 20.